Project Summary The neural circuit that controls mammalian reproduction delivers circadian information from the suprachiasmatic nucleus (SCN) via the neuropeptides arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) to the kisspeptin (Kiss) and gonadotropin-releasing hormone (GnRH) neurons, respectively. Infertility can be caused at the neuroendocrine level by genetic defects in GnRH neurons, Kiss neurons, and/or circadian pacemaker and neuropeptide neurons in the SCN. GnRH neurons arise in the olfactory placode, migrate into the hypothalamus, and extend their axons to the median eminence to deliver GnRH to the pituitary in response to Kiss signals. Disrupted migration, neurite pathfinding, and/or disregulated secretion of Kiss or GnRH neurons result in failures of puberty, fertility, and reproductive function. We have determined that the homeodomain transcription factors, Vax1, Six3, and Six6, are all crucial for reproduction. Six6 Knock-out (KO) mice exhibit strikingly decreased fertility, while heterozygote Vax1 and Six3 mice are subfertile (Vax1 and Six3 KO mice are perinatal lethal). Though GnRH neuron numbers are normal at e13.5, all three of the KO embryos have ~90% reductions in hypothalamic GnRH neurons by e17.5. Vax1, Six3, and Six6 are found in Kiss neurons and regulate expression of the Kiss promoter in Kiss-expressing cell lines. In addition, deletion of Six3 from Kiss neurons causes subfertility. Furthermore, all three KO e17.5 embryos lack SCN morphology and fail to express AVP and VIP, and Six6 KO adults lack circadian rhythms. The overall goal of this application is to elucidate the development of this fundamental reproductive neuroendocrine circuit (SCN?Kiss?GnRH) at the molecular, developmental, and physiological levels. We propose three Specific Aims: Aim 1 will investigate differentiation and maturation of GnRH neurons during migration by determining the fate of neurons lacking these factors and effects on their migratory pathway. Aim 2 will focus on the roles of Vax1, Six3, and Six6 in the development and function of Kiss neurons in vitro and in vivo with a focus on direct regulation of Kiss gene expression and effects on fertility. Aim 3 will address the fundamental mechanisms of SCN development, the contribution of Vax1, Six3, and Six6 to AVP and VIP gene expression, and investigate the roles of SCN circadian function in fertility. Our overarching hypothesis is that infertility due to defects in Vax1, Six3, and Six6 is caused by alterations in specific hypothalamic neuroendocrine cell populations within the hypothalamic SCN?Kiss?GnRH reproductive neurocircuit by regulation of development, hormone synthesis, and circadian rhythms. We believe that studies of these key homeodomain proteins will reveal fundamental mechanisms in the control of reproductive function and circadian rhythms and provide valuable insight into novel regulatory pathways important for the development and function of this neuroendocrine circuit. This multifaceted approach will yield a comprehensive understanding of their roles in fertility and circadian rhythms in vivo and in vitro, and provide further insight into the fundamental genetic control of infertility and circadian rhythms.